Temporal dissociation of COX-2-dependent arachidonic acid and 2-arachidonoylglycerol metabolism in RAW264.7 macrophages

Cyclooxygenase-2 converts arachidonic acid to prostaglandins (PGs) and the endocannabinoid, 2-arachidonoylglycerol (2-AG), to PG glyceryl esters (PG-Gs). The physiological function of PG biosynthesis has been extensively studied, but the importance of the more recently discovered PG-G synthetic pathway remains incompletely defined. This disparity is due in part to a lack of knowledge of the physiological conditions under which PG-G biosynthesis occurs. We have discovered that RAW264.7 macrophages stimulated with Kdo2-lipid A (KLA) produce primarily PGs within the first 12 h followed by robust PG-G synthesis between 12 h and 24 h. We suggest that the amount of PG-Gs quantified is less than actually synthesized, because PG-Gs are subject to a significant level of hydrolysis during the time course of synthesis. Inhibition of cytosolic phospholipase A2 by giripladib does not accelerate PG-G synthesis, suggesting the differential time course of PG and PG-G synthesis is not due to the competition between arachidonic acid and 2-AG. The late-phase PG-G formation is accompanied by an increase in the level of 2-AG and a concomitant decrease in 18:0-20:4 diacylglycerol (DAG). Inhibition of DAG lipases by KT-172 decreases the levels of 2-AG and PG-Gs, indicating that the DAG-lipase pathway is involved in delayed 2-AG metabolism/PG-G synthesis. These results demonstrate that physiologically significant levels of PG-Gs are produced by activated RAW264.7 macrophages well after the production of PGs plateaus.

Fig. S3.LC-MS/MS chromatograms of PG-G species: These representative chromatograms were obtained from analysis of cell medium collected from a plate of 10 6 RAW264.7 cells after 24 h of activation as described in Materials and Methods.Extracted PG-Gs species were analyzed on the described LC-MS/MS system in positive ion mode.The specific MRM transitions are given in Table S1, and specific chromatographic parameters are given in the table below.
Monoacylglycerols exist in an equilibrium between the 1(3)-and 2-isomer [1].The 2isomer is the initial product, and the 1(3)-isomer is a product of acyl migration.Under the analytical conditions described here, the 2-isomer elutes first, followed by the 1(3)isomer.While there is considerable chromatographic resolution between these 2 isomeric forms, our group integrates both peaks together.Fig. S4.LC-MS/MS chromatograms of arachidonic acid and arachidonoylglycerol isomers: These representative chromatograms were obtained from analysis of a cell pellet collected from a plate of 10 6 RAW264.7 cells after 24 h of activation as described in Materials and Methods.Arachidonic acid and arachidonoylglycerol isomers were analyzed on the described LC-MS/MS system in negative and positive ion mode, respectively.The specific MRM transitions are given in Table S1, and specific chromatographic parameters are given in the table below.
Similar to PG-Gs discussed in Fig. S3, arachidonoylglycerol exists as both the 1(3)-and 2-isomers, with the 2-isomer eluting first.The two isomers, which are not fully resolved, are integrated together.Thus, the 2-AG levels reported contain a minor component of 1(3)-AG.

Parameter Value
Mobile Phase A H2O + 0.1% formic acid Mobile Phase B Acetonitrile:methanol (3:1) + 0.1% formic acid Column Acquity C18 5 x 0.2 cm, 1.8 um @ 43C Gradient 65%B to 100% B in 3 min Fig.S5.LC-MS/MS chromatograms of DAG species: These representative chromatograms were obtained from analysis of a cell pellet collected from a plate of 10 6 RAW264.7 cells as described in Materials and Methods.DAG species were analyzed on the described LC-MS/MS system in positive ion mode.The specific MRM transitions are given in Table S1, and specific chromatographic parameters are given in the table below.

Fig. S7 .
Fig. S7.RAW264.7 cells were stimulated with 100 ng/mL KLA, and the designated intracellular DAGs were analyzed by LC/MS/MS.A representative experiment is shown, and the data are expressed as mean values ± S.D.

Fig. S8 .
Fig. S8.Effects of inhibitor KT-172 on extracellular PGs.RAW264.7 cells were stimulated with 100 ng/mL KLA and after 12 h of activation, different concentrations (25, 50, 100 & 250 nM) of KT-172 were added to the dish.Medium was collected after 12 h of KT-172 treatment, and extracellular levels of PGs were analyzed by LC/MS/MS.

Fig. S9 .
Fig. S9.DAGLb mRNA and protein levels: A: DAGLb protein levels at different time points (0 h, 12 h, 18 h and 24 h) were measured by western blot in cells treated with KLA along with densitometric analysis.Image J software was used to measure the intensity of the bands.B: Relative DAGLb mRNA levels normalized to GAPDH mRNA at different time points after KLA activation of RAW264.7 cells as detected by qPCR.The values are from two different experiments with each time point performed in triplicate